Hematopoiesis in mammals is maintained by a pool of self-renewing hematopoietic stem cells (HSCs) (Ogawa, 1993, Blood 81:2844-2853). HSCs feed into lineage(s)-committed undifferentiated hematopoietic progenitor cells (HPCs) with little or no self-renewal capacity (Ogawa, 1993, Blood 81:2844-2853). The HPCs in turn generate morphologically recognizable differentiated precursors and terminal cells circulating in peripheral blood.
Human HSCs are identified on the basis of their capacity for long-term hematopoietic repopulation in vitro and in vivo. Specifically, in vitro repopulation of an irradiated allogeneic stromal adherent layer in long term culture (LTC) of Dexter type has been observed. In Dexter type LTC, primitive HPCs and HSCs are assessed as five to eight week and twelve week LTC initiating cells (LTC-ICs; Sutherland et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3584-3588; Valtieri et al., 1994, Cancer Res. 54:4398-4404; Hao et al., 1996, Blood 88:3306-3313), or cobblestone area forming cells (CAFCs; Breems et al., 1996, Blood 87:5370-5378). Particularly, short term repopulating primitive HPCs have been identified in five to eight week LTC (Sutherland et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:3584-3588; Larochelle et al., 1996, Nature Med. 2:1329-1337), whereas long-term repopulating putative HSCs have been identified in twelve week LTC (Hao et al., 1996, Blood 88:3306-3313). Moreover, in vivo repopulation of severe combined immunodeficiency (SCID) mice at two months (Nolta et al., 1994, Blood 83:3041-3047) or non-obese diabetic SCID (SCID-NOD) mice at one and a half months (Bock et al., 1995, J. Exp. Med. 182:2037-2043) after irradiation and HSC injection has been observed.
In murine embryonic life (day 7.5 of gestation), a close developmental association of the hematopoietic and endothelial lineages takes place in the yolk sack blood islands, leading to the hypothesis that the two lineages share a common ancestor referred to as the hemoangioblast (Flamme et al., 1992, Development 116:435-439; Risau et al., 1995, Ann. Rev. Cell. Dev. Biol. 11:73-91).
Vascular endothelial growth factor (VEGF) and one of its receptors, VEGFRII termed Flk1 in mice and KDR in humans, play a key role in early hemoangiogenesis. In fact, Flk1xe2x88x92 knock-out mice are unable to form blood islands and blood vessels (Shalaby et al., 1995, Nature 376:62-66). Differentiated murine embryonic stem cells treated with VEGF and the ligand for c-kit receptor at the embryoid stage give rise to primitive blast cells which generate the various hematopoietic lineages (Kennedy et al., 1997, Nature 386:488-492; Kabrun et al., 1997, Development 124:2039-2048): these data suggest a role for VEGF at the level of primitive HPCs in murine embryonic hematopoiesis. There are no data concerning the effect of expression or the function of KDR in human embryonic/fetal HSCs.
In post-fetal life, the VEGF/KDR system plays an important role in the endothelial lineage. Indeed, KDR and CD34 antigens are expressed on progenitors of human adult endothelial cells (Ashara et al., 1997, Science 275:964-967). Again, there are no data concerning the effect of expression or the function of KDR in human post-fetal HSCs, particularly long-term repopulating HSCs. Most studies have focused on examination of the effect of VEGF on partially purified HPCs. The results of these studies suggest that VEGF exerts an enhancing or inhibitory effect on bone marrow (BM) HPC colony formation stimulated by diverse hematopoietic growth factors (HGFs; Broxmeyer et al., 1995, Int. J. Hematol. 62:203-215) and a stimulatory effect on hematopoietic cells in normal mice (Gabrilovich et al., 1998, Blood 92:4150-4166). In addition, KDR mRNA is expressed in cord blood (CB) and BM partially purified HPCs, while VEGF does not affect CB HPC colony formation but exerts an anti-apoptotic action on irradiated HPCs (Katoh et al., 1995, Cancer Res. 55:5687-5692).
There is a need in the art for efficient methods of purifying and characterizing long term repopulating HSCs and for methods of ex vivo expansion of these cells. In addition, there is a need in the art for methods of treating a variety of diseases using HSCs. The present invention satisfies these needs.
The invention includes a method of obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue and isolating a population of KDR+ cells therefrom, thereby obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells.
In one aspect, the human hematopoietic tissue is selected from the group consisting of embryonic hematopoietic tissue, fetal hematopoietic tissue, and post-natal henlatopoietic tissue.
In another aspect, the embryonic hematopoietic tissue is selected from the group consisting of yolk sac, and embryonic liver.
In yet another aspect, the fetal hematopoietic tissue is selected from the group consisting of fetal liver, fetal bone marrow and fetal peripheral blood.
In a further aspect, the post-natal hematopoietic tissue is selected from the group consisting of cord blood, bone marrow, normal peripheral blood, mobilized peripheral blood, hepatic hematopoietic tissue, and splenic hematopoietic tissue.
In yet a further aspect, the KDR+ cells are isolated using a reagent which specifically binds KDR.
In one aspect, the reagent is an antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.
In another aspect, the antibody is a monoclonal antibody.
In yet another aspect, the monoclonal antibody is 260.4. In a further aspect, the KDR+ cells are isolated using a conjugated vascular endothelial growth factor or a molecule derived therefrom.
In yet a further aspect, the cells are starvation resistant long-term repopulating human hematopoietic stem cells.
The invention includes an enriched population of long-term repopulating human hematopoietic stem cells obtained using a method of obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue and isolating a population of KDR+ cells therefrom, thereby obtaining a cell population enriched for long-term repopulating human hematopoietic stem cells. The invention also includes a cell isolated using this method. The invention also includes the cell isolated using this method wherein the cell comprises an isolated nucleic acid.
In one aspect, the cell comprising an isolated nucleic acid comprises an isolated nucleic acid selected from the group consisting of a nucleic acid encoding adenosine deamininase, a nucleic acid encoding xcex2-globin, a nucleic acid encoding multiple drug resistance, an antisense nucleic acid complementary to a human immunodeficiency virus nucleic acid, an antisense nucleic acid complementary to a nucleic acid encoding a cell cycle gene, and an antisense nucleic acid complementary to a nucleic acid encoding an oncogene.
In another aspect, the isolated nucleic acid is operably linked to a promoter/regulatory sequence.
In even another aspect, the promoter/regulatory sequence is selected from the group consisting of a retroviral long terminal repeat, and the cytomegalovirus immediate early promoter.
The invention includes a method of obtaining a purified population of long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue, isolating a population of hematopoietic progenitor cells therefrom, and isolating a population of KDR+ cells from the population of hematopoietic progenitor cells, thereby obtaining a purified population of long-term repopulating human hematopoietic stem cells.
In one aspect, the human hematopoietic tissue is selected from the group consisting of embryonic hematopoietic tissue, fetal hematopoietic tissue, and post-natal heniatopoietic tissue.
In another aspect, the embryonic hematopoietic tissue is selected from the group consisting of yolk sac, and embryonic liver.
In yet another aspect, the fetal hematopoietic tissue is selected from the group consisting of fetal liver, fetal bone marrow and fetal peripheral blood.
In a further aspect, the post-natal hematopoietic tissue is selected from the group consisting of cord blood, bone marrow, normal peripheral blood, mobilized peripheral blood, hepatic hematopoietic tissue, and splenic hematopoietic tissue.
In yet a further aspect, the hematopoietic progenitor cells are isolated using at least one method selected from the group consisting of isolation of cells expressing an early marker using antibodies specific for said marker, isolation of cells not expressing a late marker using antibodies specific for said late marker, isolation of cells based on a physical property of said cells, and isolation of cells based on a biochemical/biological property of said cells.
In another aspect, the early marker is selected from the group consisting of CD34, Thy-1, c-kit receptor, flt3 receptor, AC133, vascular endothelial growth factor receptor I, vascular endothelial growth factor receptor III, Tie1, Tek, and basic fibroblast growth factor receptor.
In yet another aspect, the late marker is a lineage (lin) marker.
In a further aspect, the early marker is CD34.
In even a further aspect, the hematopoietic progenitor cells are obtained from the hematopoietic tissue using an antibody which specifically binds CD34 to select a population of CD34+ hematopoietic progenitor cells.
In another aspect, the population of KDR+ cells is isolated from the population of CD34+ hematopoietic progenitor cells using an antibody which specifically binds KDR.
In yet another aspect, the antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.
In even yet another aspect, the antibody is a monoclonal antibody.
In a further aspect, the monoclonal antibody is 260.4.
In even a further aspect, the cells are starvation resistant human hematopoietic stem cells.
The invention includes an isolated purified population of long-term repopulating human hematopoietic stem cells obtained by a method of obtaining a purified population of long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue, isolating a population of hematopoietic progenitor cells therefrom, and isolating a population of KDR+ cells from the population of hematopoietic progenitor cells, thereby obtaining a purified population of long-term repopulating human hematopoietic stem cells. The invention also includes a cell obtained by this method. The invention further includes a cell obtained by this method wherein the cell comprises an isolated nucleic acid.
The one aspect, the isolated nucleic acid is selected from the group consisting of a nucleic acid encoding adenosine deaminase, a nucleic acid encoding xcex2-globin, a nucleic acid encoding multiple drug resistance, an antisense nucleic acid complementary to a human immunodeficiency virus nucleic acid, an antisense nucleic acid complementary to a nucleic acid encoding a cell cycle gene, and an antisense nucleic acid complementary to a nucleic acid encoding an oncogene.
In another aspect, the isolated nucleic acid is operably linked to a promoter/regulatory sequence.
In yet another aspect, the promoter/regulatory sequence is selected from the group consisting of a retroviral long terminal repeat, and the cytomegalovirus immediate early promoter.
In a further aspect, the hematopoietic progenitor cells are obtained from said hematopoietic tissue using antibody which specifically binds CD34 to select a population of CD34xe2x88x92 cells.
In an even further aspect, the hematopoietic progenitor cells are obtained from said population of CD34xe2x88x92 cells using antibody which specifically binds lin to select a population of CD34xe2x88x92linxe2x88x92 cells.
In another aspect, the population of KDR+ cells is isolated from the population of CD34xe2x88x92linxe2x88x92 cells using an antibody which specifically binds KDR.
In yet another aspect, the antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.
In even another aspect, the antibody is a monoclonal antibody.
In a further aspect, the monoclonal antibody is 260.4.
The invention includes an isolated purified population of long-term repopulating human hematopoietic stem cells obtained by a method of obtaining a purified population of long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue, isolating a population of hematopoietic progenitor cells therefrom, and isolating a population of KDR+ cells from the population of hematopoietic progenitor cells, thereby obtaining a purified population of long-term repopulating human hematopoietic stem cells. The invention also includes a cell obtained by this method.
The invention further includes the cell obtained by this method wherein the cell comprises an isolated nucleic acid.
In one aspect, the isolated nucleic acid is selected from the group consisting of a nucleic acid encoding adenosine deaminase, a nucleic acid encoding xcex2-globin, a nucleic acid encoding multiple drug resistance, an antisense nucleic acid complementary to a human immunodeficiency virus nucleic acid, an antisense nucleic acid complementary to a nucleic acid encoding a cell cycle gene, and an antisense nucleic acid complementary to a nucleic acid encoding an oncogene.
In another aspect, the isolated nucleic acid is operably linked to a promoter/regulatory sequence.
In yet another aspect, the promoter/regulatory sequence is selected from the group consisting of a retroviral long terminal repeat, and the cytomegalovirus immediate early promoter.
The invention includes a method of expanding a population of long-term repopulating human hematopoietic stem cells. The method comprises obtaining a population of cells from human hematopoietic tissue, isolating a population of KDR+ hematopoietic stem cells therefrom, and incubating the population of KDR+ cells with vascula endothelial growth factor, thereby expanding the population of long-term repoputating human hematopoietic stem cells.
In one aspect, the method further comprises incubating the population of KDR+ cells with at least one growth factor.
In another aspect, the growth factor is selected from the group consisting of flt3 receptor ligand, kit receptor ligand, thrombopoietin, basic fibroblast growth factor, interleukin 6, interleukin 11, interleukin 3, granulomonocytic colony-stimulatory factor, granulocytic colony-stimulatory factor, monocytic colony-stimulatory factor, erythropoietin, angiopoietin, and hepatocyte growth factor.
The invention also includes an isolated purified population of long-term repopulating human hematopoietic stem cells obtained by this method.
The invention further includes a cell obtained using this method.
In one aspect, the cell comprises an isolated nucleic acid.
In another aspect, the isolated nucleic acid is selected from the group consisting of a nucleic acid encoding adenosine deamninase, a nucleic acid encoding xcex2-globin, a nucleic acid encoding multiple drug resistance, an antisense nucleic acid complementary to a human immunodeficiency virus nucleic acid, an antisense nucleic acid complementary to a nucleic acid encoding a cell cycle gene, and an antisense nucleic acid complementary to a nucleic acid encoding an oncogene.
In yet another aspect, the isolated nucleic acid is operably linked to a promoter/regulatory sequence.
In a further aspect, the promoter/regulatory sequence is selected from the group consisting of a retroviral long terminal repeat, and the cytomegalovirus immediate early promoter.
The invention includes a blood substitute comprising the progeny cells of an isolated purified population of long term repopulating human hematopoietic stem cells.
In one aspect, the progeny cells are selected from the group consisting of red blood cells, neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes, monocytes, dendritic cells, platelets, B lymphocytes, T lymphocytes, natural killer cells, and differentiated precursors thereof, and undifferentiated progenitors thereof.
The invention also includes a chimeric non-human mammal comprising at least one of an isolated and purified long-term repopulating human hematopoietic stem cell.
In one aspect, the cell is introduced into the mammal using a method selected from the group consisting of transplantation, and blastocyst injection.
In another aspect, the mammal is selected from the group consisting of a mouse, a rat, a dog, a donkey, a sheep, a pig, a horse, a cow, a non-human primate.
The invention includes a method of inhibiting rejection of a transplanted organ. The method comprises ablating the bone marrow of a transplant recipient and administering to the recipient a multi-lineage engrafting dose of an isolated and purified long-term repopulating human hematopoietic stem cell obtained from the hematopoietic tissue of the donor of said organ, thereby inhibiting rejection of a transplanted organ.
The invention includes a method of transplanting an autologous human hematopoietic stem cell in a human. The method comprises obtaining a population of cells from the hematopoietic tissue of a human and isolating a population of non-malignant hematopoietic stem cells therefrom, ablating the bone marrow of the human, and administering at least one isolated non-malignant hematopoietic stem cell to the human, thereby transplanting an autologous human hematopoietic stem cell in a human.
The invention also includes a method of isolating a KDR+ cell. The method comprises selecting a cell expressing an antigen coexpressed with KDR, thereby isolating a KDR+ cell.
In one aspect, the coexpressed antigen is selected from the group consisting of a vascular endothelial growth factor receptor I, and a vascular endothelial growth factor receptor III.
The invention includes a method of isolating a KDR+ stem cell giving rise to at least one of a muscle cell, a hepatic oval cell, a bone cell, a cartilage cell, a fat cell, a tendon cell, and a marrow stroma cell. The method comprises isolating a KDR+ stem cell from hematopoietic tissue, thereby isolating a KDR+ stem cell giving rise to at least one of a muscle cell, a hepatic oval cell, a bone cell, a cartilage cell, a fat cell, a tendon cell, and a marrow stroma cell.
The invention includes a method of monitoring the presence of KDR+ stem cells in a human hematopoietic tissue in a human receiving therapy. The method comprises obtaining a sample of hematopoietic tissue from the human before, during and after the therapy, and measuring the number of KDR+ stem cells in the sample, thereby monitoring the presence of KDR+ stem cells in a human hematopoietic tissue obtained from a human receiving therapy.